Accumulation of hepatic lipids either as a result of obesity or lipodystrophy has been linked to the development of hepatic insulin resistance. Particularly, in obese individuals chronically elevated serum free fatty acids (FFA) and high insulin levels lead to increased FFA uptake by the liver and increased synthesis of lipids resulting in hepatic steatosis. Here we postulate that the sustained protein-mediated uptake and activation of FFAs by the liver is required for obesity-associated hepatic steatosis and insulin resistance. Thus inhibition of these key proteins should prevent hepatic TG accumulation. Importantly, a reversal of hepatic steatosis might restore hepatic, and potentially whole body, insulin sensitivity. We identified as key targets for these studies the two predominant hepatic fatty acid transport proteins, FATP2 and FATP5 (Slc27a2 and -a5) and two long-chain acyl CoA synthetases (ACSL1 and -4) that are thought to be crucial for the accumulation of hepatic lipids. Following a characterization of lipid fluxes facilitated by these four proteins we will down-regulate their function in three model systems of hepatic steatosis, i.e. high-fat nutrient overload, hyperphagy (ob/ob mice), and lipoathrophy (A-ZIP/F-1 mice) to test whether loss of FATP2/5 or ACSL1/4 function can reverse hepatic steatosis. Once we have established reversal of hepatic steatosis we will determine the impact on hepatic insulin sensitivity and glucose homeostasis. In summary, these studies will further elucidate the connections between obesity associated hepatic TG accumulation and insulin resistance by utilizing an innovative approach for the suppression of endogenous liver genes and may lead to novel approaches for the treatment of hepatic steatosis and diabetes.